European Journal of Clinical Microbiology & Infectious Diseases (2023) 42:1125–1133

https://doi.org/10.1007/s10096-023-04646-1

ORIGINAL ARTICLE

Phenotypic and genomic characteristics of oxacillin‑susceptible

mecA‑positive Staphylococcus aureus, rapid selection of high‑level
resistance to beta‑lactams
Vladimir Gostev1,2 · Ksenia Sabinova1 · Julia Sopova3,4 · Olga Kalinogorskaya1 · Ofeliia Sulian1 · Polina Chulkova1 ·
Maria Velizhanina4,5 · Polina Pavlova1,3 · Lavrentii Danilov3 · Lyudmila Kraeva6 · Dmitrii Polev6 · Elvira Martens1,2 ·
Sergey Sidorenko1,2

Received: 10 April 2023 / Accepted: 20 July 2023 / Published online: 29 July 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
The aim of this study is to describe the phenotypic and genetic properties of oxacillin-susceptible methicillin-resistant
Staphylococcus aureus (OS-MRSA) isolates and their beta-lactam resistant derivatives obtained after selection with oxacillin.
A collection of hospital- (HA-) and community-acquired (CA-) MRSA was screened for oxacillin susceptibility. Antibiotic
susceptibility testing, population analysis profile (PAP), mecA expression analysis, and whole genome sequencing (WGS)
were performed for 60 mecA-positive OS-MRSA isolates. Twelve high-level beta-lactam resistant derivatives selected during
PAP were also subjected to WGS. OS-MRSA were more prevalent among CA-MRSA (49/205, 24%) than among HA-MRSA
(11/575, 2%). OS-MRSA isolates belonged to twelve sequence types (ST), with a predominance of ST22-t223-SCCmec IVc
and ST59-t1950-SCCmec V lineages. OS-MRSA were characterized by mecA promoter mutations at − 33 (C→T) or − 7
(G→T/A) along with PBP2a substitutions (S225R or E246G). The basal and oxacillin-induced levels of mecA expression in
OS-MRSA isolates were significantly lower than those in control ST8-HA-MRSA isolates. Most of the OS-MRSA isolates
were heteroresistant to oxacillin. High-level beta-lactam resistant OS-MRSA derivatives selected with oxacillin carried muta-
tions in mecA auxiliary factors: relA (metabolism of purines), tyrS, cysS (metabolism of tRNAs), aroK, cysE (metabolism of
amino acids and glycolysis). Cefoxitin-based tests demonstrated high specificity for OS-MRSA detection. The highest positive
predictive values (PPV > 0.95) were observed for broth microdilution, the VITEK® 2 automatic system, and chromogenic
media. Susceptibility testing of CA-MRSA requires special attention due to the high prevalence of difficult-to-detect OS-
MRSA among them. Mis-prescription of beta-lactams for the treatment of OS-MRSA may lead to selection of high-level
resistance and treatment failures.

Keywords Staphylococcus aureus · MRSA · OS-MRSA · Penicillin–clavulanate · Oxacillin · Cefoxitin · Antimicrobial

resistance selection

Introduction is recognized as a difficult-to-detect pathogen [3] and the

In 1992, Hiramatsu et al. described mecA-positive “Border-
line-resistant Methicillin-resistant Staphylococcus aureus”
isolates that demonstrated low-level methicillin MICs [1].
In the early 2000s, such phenotypes were described in more
detail and defined as oxacillin-susceptible methicillin-resist-
ant Staphylococcus aureus (OS-MRSA) [2]. OS-MRSA
* Sergey Sidorenko
sidorserg@gmail.com
Extended author information available on the last page of the article
main concern is reporting false susceptibility to beta-lac-
tams, even when using cefoxitin-based tests, which could
result in inappropriate antibiotic prescribing [4]. At pre-
sent, OS-MRSA isolates have been reported in geographi-
cally distinct regions: the USA [5, 6], Brazil [7], the UK
[8, 9], Europe [10, 11], Africa [12], Iran [13], India [14],
and China [15–17]. Recently, OS-MRSA was isolated from
pets [18], other animal species [19], and food [20–22]. The
population of OS-MRSA is diverse and is represented by
different genetic lineages. This feature is more attributable
to the methicillin-susceptible S. aureus (MSSA) population
than to MRSA. However, in China and Taiwan, the most

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1126 European Journal of Clinical Microbiology & Infectious Diseases (2023) 42:1125–1133
predominant OS-MRSA clone is sequence type (ST) ST59,
and in African countries ST88. In the other studies, OS-
MRSA genetic lineages represented by different clones—
ST1, ST2, ST5, ST8, ST121, ST89, ST22, ST45, and ST30.
In the majority of cases, OS-MRSA harbored staphylococcal
cassette chromosome mec (SCCmec) types IV and V. It has
been suggested that OS-MRSA may represent a stage of the
phenotypic evolution of MRSA from «pre-MRSA» [23].
Mutations in the promoter region of mecA play a cru-
cial role in susceptibility to oxacillin [24, 25]. Mutations in
blaR1-blaI of the bla operon [26, 27], acquisition of stop
codons, and frameshift mutations in mecA have also been
described in mecA-positive oxacillin-susceptible isolates [5].
Control of mecA expression is dependent on many auxil-
iary factors that determine methicillin resistance in the core
genome of S. aureus [28]. In the recent study of Boonsiri
et al. [29], it was found that expression of oxacillin resist-
ance in OS-MRSA was strongly related to the metabolic
changes mediating a stringent-like response. Unexpected
susceptibility to the penicillin–clavulanate combination was
observed in mecC- and mecA-positive isolates in the studies
of Ba et al. [30] and Harrison et al. [24], respectively. This
unusual phenotype results from simultaneous mutations in
mecA and its promoter. Treatment with penicillin–clavula-
nate in a staphylococcal wax moth larvae infection model
has shown promising results [24]. However, the question
of the possibility of using early penicillins and their com-
bination with beta-lactamase inhibitors for the treatment of
OS-MRSA infections has not been finally resolved, in vitro
beta-lactams can induce high levels of resistance in OS-
MRSA [29].
Here, we describe phenotypic and genetic properties of
diverse OS-MRSA isolates, collected during different time
periods, and rapid selection of beta-lactam resistance after
short-term exposure to oxacillin.
Material and methods
Ethics approval and consent to publication
Not required. Only bacterial isolates were subjected to the
study. MRSA isolates were collected in participating centers
along with record forms. Personal data of patients was not
included in record forms.
Bacterial isolates
Hospital-associated (HA)-MRSA (n = 575), isolated from
hospitalized patients with nosocomial staphylococcal
infections, and community-associated (CA)-MRSA (n =
205) were included in the study; CA-MRSA was isolated
from healthy adults and children during routine screening
13
for staphylococcal carriage between 2011 and 2019 in 11
cities and 30 medical centers of the Russian Federation.
In addition, 21 OS-MRSA isolates from a previous study
were included [31]. Five HA-MRSA isolates belonging
to ST8-t008-SCCmec IVc were used as control strains for
the broth microdilution (BMD) tests, population analysis
profile (PAP), and mecA expression assays.
OS‑MRSA detection
All isolates were tested for the presence of mecA using
PCR with the following primers: forward: 5′-AAG​T TT​
GCA​TAA​GAT​CTA​TAA-3′, reverse: 5′-ATT​TAT​GTA​TGG​
CAT​GAG​TAA-3′. PCR was performed with DreamTaq
PCR Master Mix (ThermoFisher Scientific, USA) in a
volume of 25 μl. Amplification was carried out accord-
ing to the following protocol: denaturation: 95 °C, 5 min;
35 cycles: 95 °C, 10 s; 50 °C, 10 s; final elongation at 72
°C, 5 min. Amplicons (672 bp) were visualized by gel
electrophoresis. Susceptibility to cefoxitin and oxacillin
was evaluated using several approaches: the disc-diffusion
method (DDM) with cefoxitin (30 μg) and oxacillin (1
μg) disks (Bio-Rad); the gradient diffusion method (GDM;
ETEST®, bioMérieux); the VITEK® 2 compact system
with AST-GP67 cards (bioMérieux); and a chromogenic
medium (CM, Brilliance MRSA 2 Agar®; Oxoid, UK). In
addition, Mueller–Hinton agar (Bio-Rad) supplemented
with 2% NaCl was used for the DDM and GDM tests. The
minimal inhibitory concentrations (MICs) of beta-lactams
and other antibiotics (Molekula, UK) were determined
using the BMD method, according to ISO 20776-1-2010,
and interpreted according to the European Committee on
Antimicrobial Susceptibility Testing (EUCAST) recom-
mendations; for the oxacillin DDM, a breakpoint of R ≤
10 mm was used. OS-MRSA was defined as mecA-positive
with susceptibility to oxacillin using the BMD method.
Population analysis profile with oxacillin
PAP was performed according to the microdilution modi-
fication described in a previous study [32]. Four 10-fold
dilutions of the initial suspension ­(108 CFU/mL) for each
strain were prepared. Three 10 μL droplets of each dilution
were plated onto oxacillin-containing Brain-Heart infu-
sion (BHI) agar plates (0.06–32 mg/L). The inoculated
plates were incubated for 48 h at 37 °C. Plated droplets
containing 5–50 CFUs were selected for counting, and the
average number of colonies per oxacillin concentration
was determined. Plots showing the number of CFUs per
oxacillin concentration were prepared by calculating the
area under the curve (AUC).
European Journal of Clinical Microbiology & Infectious Diseases (2023) 42:1125–1133
mecA expression assay
mecA expression was analyzed using RT-PCR in BHI broth
(Bio-Rad), and induction was achieved by the addition of
0.016 mg/L of oxacillin. Samples were collected at the late-
exponential phase of growth and incubated with RNAprotect
reagent (QIAGEN, Germany). Prior to RNA extraction, bac-
terial cells were mechanically disrupted in a cool environ-
ment (4 °C) for 30 min using glass beads (Sigma-Aldrich,
USA). Total RNA was extracted and purified using the
PureLink™ RNA Mini Kit (ThermoFisher Scientific, USA).
DNase treatment and reverse transcription were performed
using a cDNA Synthesis Kit (K1681; ThermoFisher Scien-
tific, USA). The probes mecA (target) and nuc, which codes
the thermonuclease protein (reference internal control),
labelled with FAM and ROX dyes, respectively, were used
in a single multiplex PCR (probes and primer sequences
are presented in Supplementary Table S1). Eight OS-MRSA
isolates and one control (HA-MRSA-ST8) were included in
the experiment. Measurements were performed in triplicate.
Expression fold-changes were calculated using the ­2-∆∆Ct
method [33].
Whole genome sequencing
A Nextera Flex Kit (Illumina, San Diego, CA) was used
for DNA library preparation, followed by sample indexing
and amplification, according to the manufacturer’s protocol.
The DNA libraries were sequenced using MiSeq (Illumina,
San Diego, CA). Genomic data have been deposited in the
NCBI Sequence Read Archive (SRA), under BioProject
PRJNA872007.
Bioinformatics and statistical analysis
After reads filtering and trimming the contigs were de novo
assembled. Phylogenetic analysis, molecular typing, and sin-
gle-nucleotide polymorphisms (SNP) calling and annotation
were performed with Snippy (https://​github.​com/​tseem​ann/​
snippy), mlst (https://​github.​com/t​ seem​ann/​mlst), ABRicate
(https://​github.​com/​tseem​ann/​abric​ate), respectively. Statis-
tical analyses were performed in R using the Mann–Whitney
U test and Spearman’s correlation analysis with the corrplot
package (https://​cran.r-​proje​ct.​org/​web/​packa​ges/​corrp​lot).
Results
OS‑MRSA characterization
Among the 575 HA-MRSA and 205 CA-MRSA isolates,
11 (2%) and 49 (24%) OS-MRSA were detected, respec-
tively. Of the 60 OS-MRSA isolates (Table 1), 21 (35%)
1127
belonged to ST22 (Gaza Strip clone), and most of them
were drawn from healthy carriers. Ten isolates (17%)
belonged to the ST59 clone; nine were from healthy car-
riers and one from a hospital-acquired skin and soft tissue
infection (SSTI). The remaining 29 isolates belonged to
ten different STs; 20 were from healthy carriers and nine
HA-MRSA from patients with SSTI, osteomyelitis, and
bacteremia. The complete characterization of the geno-
types and phenotypes of the isolates is presented in Sup-
plementary Fig. S1 and Supplementary Table S2.
OS-MRSA demonstrated a low level of resistance to
non-beta-lactam antibiotics, and, on average, each isolate
was resistant to two groups of antibiotics. Resistance to
macrolides was mediated by ermB and ermC; to tetracy-
clines, by tetK and tetM; to phenicols, by cat and fexA;
to fusidic acid, by fusC; to aminoglycosides, by aac(6')-
aph(2”), ant(6)-Ia, and aph(3')-III; and mutations in gyrA
(S84L) and parC (S80F) mediated resistance to fluoro-
quinolones. All isolates were susceptible to vancomycin,
daptomycin, linezolid, tigecycline, and ceftaroline. The
presence of virulence genes has been associated with spe-
cific STs; lukFS (PVL) was identified in ST2704 isolates,
tsst in ST22, and seb in ST59.
All isolates included in the study demonstrated an oxacil-
lin MIC of ≤ 2.0 mg/L, determined using BMD. The isolates
had high MIC values of penicillin ­(MIC90 8 mg/L), amoxi-
cillin ­(MIC90 64 mg/L), and cefoxitin (­ MIC90 16 mg/L), but
meropenem MIC did not exceed 2.0 mg/L. When clavula-
nate was added to the penicillins, a decrease in MICs was
detected (penicillin ­MIC90, 2.0 mg/L; amoxicillin ­MIC90,
16 mg/L) in blaZ-positive isolates (Table 1). The bla genes
(blaZ, blaR1, and blaI) were carried by plasmids, except
all ST22 and ST1 isolates, wherein blaZ was located on the
chromosome. We noted no differences in the phenotypes
between isolates with plasmids and the chromosomal locali-
zation of blaZ. The OS-MRSA phenotypes were significantly
different from those of typical MRSA (HA-MRSA), which
were characterized by high MIC values for both penicillins
and meropenem and a lack of synergism between penicillins
and clavulanate (Supplementary Table S2).
OS-MRSA harbored SCCmec types IV and V. Most
ST22 isolates (17 of 21) harbored the wild type mecA gene.
The remaining ST22 isolates and those of other STs car-
ried E246G amino acid substitutions (AAS) and different
combinations of five additional AAS in PBP2a. We did not
detect any insertions, deletions, or stop codons. In all OS-
MRSA, we identified SNPs in the mecA promoter (Table 1)
at positions − 33 (C→T) or − 7 (G→T/A). We did not
detect any isolates harboring both substitutions simultane-
ously. In one isolate, a mutation at − 38 (A→G) was found
in addition to the substitution in position − 7 (G→T/A),
the isolate was blaZ-negative and demonstrated penicillin
MIC 8.0 mg/L. This genotype has been associated with
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1128 European Journal of Clinical Microbiology & Infectious Diseases (2023) 42:1125–1133

Table 1  Genotypes and mutations associated with oxacillin susceptibility in mecA-positive S. aureus
MLST, ST Spa SCCmec N blaZ mecA promoter PBP2a MIC, mg/L
− 38 − 33 − 7 S225R A228T E246G D323N A468V S590P PEN PEN-CL

ST22 t223 IVa, IVc 17 + − − + − − - − − − 1–8 0.5–4

ST22 t223 IVa, IVc 3 +/- (1) * − − + − − + − − − 1–8 0.5–2
ST22 t223 IVc 1 - − − + − − + + − − 0.5 0.5
ST59 t1950 Vb 10 +/- (2) − + − + − + − − − 0.125–2 0.25–0.5
ST1 t321 IVa 5 + − − + − − + − − − 2–4 0.5–1
ST1 t127 IVa 1 + − − + − − + − − − 4 0.5
ST8 t008 IVc 3 + − − + − − + − − − 8–32 1–2
ST8 t008 IVc 1 - + − + − − + − − − 8 8
ST8 t3308 V 1 + − + − + − + − − − 8 0.5
ST5 t688 V 5 +/- (1) − + − + − + − − + 0.5–4 0.25–0.5
ST2704 t002 IVc 5 + − − + − − + − − − 2–8 0.5–1
ST97 t359 IVb 1 + − − + − − + − − − 8 2
ST97 t9129 V 1 + − + − + − + − − − 16 1
ST6 t1950 V 1 + − + − + − + − − − 1 0.25
ST6 t304 IVa 1 + − − + − − + − − − 8 2
ST45 NT Vc 1 + − + − + − + − − − 16 0.25
ST398 t034 Vc 1 - − + − + + + − − − 0.25 0.125
ST1535 t084 V 1 + − + − + − + − − − 1 0.5
ST7 t091 IVa 1 + − − + − − + − − − 16 4

Most prevalent spa and SCCmec types are shown

*In brackets, number of blaZ-negative isolates, PEN penicillin, PEN–CL penicillin-clavulanic acid

penicillin–clavulanate resistance, and similar results were
obtained by Chen et al. [34].
The basal level expression of mecA in OS-MRSA was
extremely low compared with that in the control ST8-HA-
MRSA isolate (p < 0.05) (Supplementary Fig. S2). Induc-
tion by oxacillin increased the level of mecA transcripts in
both control and OS-MRSA isolates, but the level of expres-
sion in the control isolate remained significantly higher (p <
0.05). The isolates with the mecA promoter mutation at posi-
tion − 33 demonstrated lower levels of basal and induced
mecA expression compared with that of isolates with muta-
tion at position – 7; however, the difference was not statisti-
cally significant (p = 0.3).
PAP analysis and resistance selection
PAP analysis showed that most OS-MRSA isolates were
represented by heterogeneous populations. Isolates with a
promoter mutation at position − 7 demonstrated higher AUC
values than those of mecA-negative isolates, and lower AUC
values than those of the control mecA-positive HA-MRSA
(Supplementary Fig. S3). Isolates with a promoter mutation
at position − 33 were represented by two groups (Supple-
mentary Fig. S3). One group did not differ substantially from
mecA-negative isolates (MSSA-like), while the other showed
a slight increase in AUC.
Among the 60 isolates subjected to PAP analysis, 21
demonstrated growth of single colonies in the presence of
32.0 mg/L of oxacillin. When these colonies were subcul-
tured on fresh medium with 32.0 mg/L of oxacillin, the
growth was obtained only in 12 cases after 48 h of incu-
bation (Fig. 1A). These derivative strains were subjected
to more detailed phenotypic testing and WGS. Derivative
isolates showed a decrease in growth parameters (Fig. 1C),
lag phase was increased from 104 min (IQR: 90–129) for
parental strains to 178 min (IQR: 138–198). However, the
median doubling time of derivative strains did not differ
significantly from that of the parental strains (33 min and
31 min, respectively). Increased beta-lactam MICs were
detected in most derivative strains; oxacillin and peni-
cillin-clavulanate MICs ranged from 2 to 32 mg/L and 2
to 8 mg/L, respectively (Supplementary Table S4). The
majority of SNPs in derivative strains were either non-syn-
onymous in genes involved in the metabolism of purines
(relA, gmk), tRNAs (tyrS, cysS), amino acids (aroK, cysE,
serA), and glycolysis (eno, pyk, fbaA) or located in the
upstream regions of mentioned genes (Fig. 1B, Supple-
mentary Table S3). No significant difference was found
in basal and induced expression levels of mecA between
parental and derivative isolates, except for two derivative
isolates (Fig. 1B, Supplementary Fig. S4). The derivatives
SA1161 and SA1205 were characterized by significantly

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European Journal of Clinical Microbiology & Infectious Diseases (2023) 42:1125–1133
Fig. 1  Genotypic and phenotypic features of the OS-MRSA derivatives.
A Scheme of selection for OS-MRSA derivative strains; B SNPs, mecA
expression and MICs comparison of the OS-MRSA and its derivative
increased levels of mecA transcripts by 100- and 10-fold
(p < 0.001), respectively.
Comparative analysis of antimicrobial susceptibility
tests for OS‑MRSA detection
A comparative analysis of different antimicrobial suscep-
tibility tests (AST) showed that cefoxitin-based tests are
highly specific for the detection of mecA-positive pheno-
types. In particular, the highest positive predictive values
(PPV > 0.95) were obtained using BMD, the VITEK® 2
automatic system, and chromogenic media (Table 2). Adding
2% sodium chloride to the media significantly increased PPV
(up to 0.92) in oxacillin-based tests; sodium chloride has
1129
strains; C Growth rate comparison. FOX cefoxitin, OXA oxacillin, CPT
ceftaroline, PEN penicillin, PEN-CL penicillin-clavulanic acid, AMX
amoxicillin, AMX-CL amoxicillin-clavulanic acid, MER meropenem
already been used for OS-MRSA detection [35]. Using cor-
relation analysis, the correlation between different methods
for methicillin detection was not found for all tests (Supple-
mentary Fig. S5A). In particular, cefoxitin BMD data were
not correlated with cefoxitin-based DDM, GDM, or GDM
with oxacillin tests. The correlation between the level of pen-
icillin–clavulanate MICs and other antibiotic susceptibility
tests was either low or absent. We observed a tendency (p =
0.044) for promoter mutation positions to influence the dis-
tribution of inhibition zones in cefoxitin DDM (Supplemen-
tary Fig. S5B). For isolates with a mutation at position − 33,
the diameters of inhibition zones were in the range of 17–22
mm, whereas in isolates with a mutation at − 7, values were
more widely distributed (14–25 mm).
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1130 European Journal of Clinical Microbiology & Infectious Diseases (2023) 42:1125–1133

Table 2  Comparison of Tests Sa (n) Ra (n) PPV MIC (mg/L) or DDM (mm) MIC50 MIC90
different antibiotic susceptibility
tests for detection of the Range Geo. mean
methicillin-resistant phenotype
in mecA-positive S. aureus OXA BMD 60 0 0 0.25–2 0.7 0.5 2
OXA GDM 49 11 0.18 0.5–24 1.6 1.5 4
VITEK® 2 OXA 53 7 0.11 ≤ 0.25–> 4 NA NA NA
OXA ­DDMb 41 19 0.32 0–23 14 NA NA
OXA GDM Salt 13 47 0.78 1–256 4.7 4 16
FOX GDM 12 48 0.8 3–64 9.7 8 16
FOX GDM Salt 10 50 0.83 4–48 9.1 8 16
FOX DDM 7 53 0.9 0–25 18 NA NA
OXA DDM S­ altb 5 55 0.92 0–19 0 NA NA
FOX DDM Salt 5 55 0.92 8–24 18.5 NA NA
FOX BMD 2 58 0.96 2–32 11 8 16
CM 2 58 0.96 NA NA NA NA
VITEK® 2 FOX 1 59 0.98 NA NA NA NA

Geo. mean geometric mean, NA not applicable, PPV positive predictive value
a
According to EUCAST 2022 breakpoints
b
Breakpoint R ≤ 10 mm

Discussion of promoter mutations in reduction of mecA expression [24,

OS-MRSA requires special attention because this phenotype
may be associated with inappropriate use of beta-lactams
and treatment failures. Currently, resistance to cefoxitin is
considered a reliable (although not 100% sensitive and spe-
cific) marker of the presence of the mecA gene and clini-
cal resistance to all beta-lactams. However, the problem of
mecA-positive staphylococci resistance to beta-lactams is
more complex. Early penicillins show greater affinity for
PBP2a and lower MICs in comparison with cephalosporins.
There is also evidence of successful treatment of MRSA
experimental infections and limited data on the clinical effi-
cacy of penicillins [36].
Using a collection of MRSA isolates with different levels
of susceptibility to oxacillin, Harrison et al. [24] demon-
strated that penicillin susceptibility in blaZ-negative MRSA
and penicillin-clavulanate susceptibility in blaZ-positive
MRSA is mediated by a combination of two different muta-
tions in the mecA promoter region (at positions − 7 or − 33)
and by either one of two substitutions in PBP2a (E246G or
M122I). In our study, only OS-MRSA with low oxacillin and
meropenem MICs were included. They originated mainly
from healthy carriers and belonged to two lineages of CA-
MRSA, ST22 and ST59. Both lineages are found among
healthy carriers in the Middle East [31, 37] and China [15].
Isolates of both lineages carried the same mutations in mecA
and mecA promoter regions, showed low MIC values of early
penicillins when combined with beta-lactamase inhibitors,
and demonstrated a low level of basal and oxacillin-induced
mecA expression. Previous allele replacement experiments
with wild-type MRSA ST22 and ST59 confirmed the role
34]. An important feature of OS-MRSA was heteroresistance
to oxacillin, most pronounced in isolates with mutations in
position − 7 of the promoter region.
We observed that exposure of OS-MRSA to oxacillin
during PAP analysis leads to the selection of a high level of
beta-lactam resistance associated with mutations in pathways
of glycolysis (pyk, fbaA), purines (gmk), alarmone synthesis
(relA), and amino acid metabolism (tyrS, cysS, cysE, serA),
but we did not detect mutations directly associated with
beta-lactam resistance. The identified mutations are linked
to auxiliary factors, which regulate the level of beta-lactam
resistance in MRSA [28]. Notably, SNPs in mecA and its
promoter were still present in every isolate after selection.
Similar results were obtained in the recent study of Boonsiri
et al. [29]; it was found that expression of oxacillin resistance
was strongly related to the following genes: RNA polymer-
ase (rpoBC), purine biosynthesis (guaA, prs, hprT), alarmone
synthesis (relA), glycolysis (pykA, fbaA, fruB), protein quality
control (clpXP, ftsH), and tRNA synthase (lysS, gltX). The
stringent stress response induced by the alarmone synthe-
sis is involved in the beta-lactam resistance of MRSA. The
role of relA is well-established in this process [38]. Other
metabolic changes, including: purine biosynthesis, glycolysis,
and tRNA synthesis are could also contribute to beta-lactam
resistance [38–40]. Moreover, in the current study was shown
that most of derivative strains with high MICs to beta-lactams
did not confer increasing of level of mecA expression. This
finding suggests that beta-lactam resistance depends not only
on the functionality of mec/bla operons but also on meta-
bolic changes. As was shown in the study of Boonsiri et al.
[29] despite the increase of oxacillin MIC, PBP2a production

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European Journal of Clinical Microbiology & Infectious Diseases (2023) 42:1125–1133
and mecA expression were not significantly changed in OS-
MRSA derivatives. Our results agreed with conception of
stringent-like response which might direct bacterial metabo-
lism toward the acquisition of oxacillin resistance [29].
Our data on the high heterogeneity of OS-MRSA suggest
that the rapid selection of resistant derivatives is associ-
ated with the preexistence of a minor subpopulation in the
general population rather than with the selection of newly
emerged mutations.
In the present study, comparative analysis showed dis-
cordance between different phenotypic methods for MRSA
detection. The interpretations of diffusion method results did
not match those of the dilution method. Direct detection of
the mecA gene is the preferred method for detecting methi-
cillin resistance among CA-MRSA. Susceptibility testing of
CA-MRSA isolates requires a particularly careful approach
since OS-MRSA is widespread among them. The presence
of a subpopulation with high MICs threatens the transforma-
tion of OS-MRSA into MRSA, with a high level of resist-
ance to beta-lactams, and questions the use of beta-lactams
for the treatment of OS-MRSA infections.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 10096-0​ 23-0​ 4646-1.
Author contributions Conceptualisation and design: Vladimir Gostev,
Sergey Sidorenko and Julia Sopova. Material preparation, data col-
lection and analysis were performed by Olga Kalinogorskaya, Ofeliia
Sulian, Polina Chulkova, Lavrentii Danilov, Lyudmila Kraeva, Dmitrii
Polev and Elvira Martens. Genome editing: Ksenia Sabinova, Maria
Velizhanina and Julia Sopova. Bioinformatics: Polina Pavlova.The first
draft of the manuscript was written by Vladimir Gostev and all authors
commented on previous versions of the manuscript. All authors read
and approved the final manuscript.
Funding Genomic, phenotypic, and bioinformatic analysis was funded
by the Russian Science Foundation (grant no. 18-75-10114-P). Gene
expression experiments was supported by the project ID 94030690 of
the St. Petersburg State University, St. Petersburg, Russia.
Data availability Genomic data have been deposited in the NCBI
Sequence Read Archive (SRA), under BioProject PRJNA872007.
Declarations
Ethics approval Not required.
Consent to participate Not applicable.
Consent for publication Not applicable.
Competing interests The authors declare no competing interests.
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European Journal of Clinical Microbiology & Infectious Diseases (2023) 42:1125–1133 1133

Authors and Affiliations

Vladimir Gostev1,2 · Ksenia Sabinova1 · Julia Sopova3,4 · Olga Kalinogorskaya1 · Ofeliia Sulian1 · Polina Chulkova1 ·
Maria Velizhanina4,5 · Polina Pavlova1,3 · Lavrentii Danilov3 · Lyudmila Kraeva6 · Dmitrii Polev6 · Elvira Martens1,2 ·
Sergey Sidorenko1,2

Vladimir Gostev Dmitrii Polev

guestvv11@gmail.com polev@pasteurorg.ru
Ksenia Sabinova Elvira Martens
xennia55@gmail.com eamartens@yandex.ru
Julia Sopova 1
Pediatric Research and Clinical Center for Infectious
sopova@hotmail.com
Diseases, Professor Popov Str. 9, Saint Petersburg 197022,
Olga Kalinogorskaya Russia
kalinogorskaya@bk.ru 2
North-Western State Medical University Named
Ofeliia Sulian After I. I. Mechnikov, Piskarevskij Prospect 47,
sulyan1994@mail.ru Saint Petersburg 195067, Russia
3
Polina Chulkova Saint Petersburg State University, Universitetskaya
sps_96@mail.ru Embankment, Saint Petersburg, 7‑9, 199034, Russia
4
Maria Velizhanina Vavilov Institute of General Genetics, Universitetskaya
velizhanina.me@gmail.com Embankment 7-9, Saint Petersburg 199034, Russia
5
Polina Pavlova All-Russia Research Institute for Agricultural Microbiology,
pollypavlova98@gmail.com Podbelsky Chausse 3, Saint Petersburg, Pushkin 8, 196608,
Russia
Lavrentii Danilov
6
lavrentydanilov@gmail.com Saint Petersburg Pasteur Institute, Mira Str.14,
Saint Petersburg 197101, Russia
Lyudmila Kraeva
kraeva@pasteurorg.ru

13